Total synthesis of human ACTH. The synthesis was carried out by the solid phase method in the Beckman Model 990 Peptide Synthesizer. From 3.23 g N alpha-Boc-gamma-B21-Glu-Phe-resin, 5.61 g of the fully protected alpha h-ACTH-resin was obtained. The identity of the synthetic product with the natural hormone was established by amino acid analysis, chromatography on CMC, partition chromatography on Sephadex G-50, optical rotation, CD spectra, electrophoresis on both paper and polyacrylamide, electrophoretic patterns of both chymotryptic and tryptic digests, as well as by three biological tests. Inhibition of ACTH induced cAMP synthesis in isolated rat adrenal cells by NPS-ACTH. Chemical modification of the single tryptophan residue in ACTH by reaction with o-nitrophenyl sulfenyl chloride results in the loss of the lipolytic activity of the hormone in isolated rat fat cells. Moreover, the o-nitrophenyl sulfenyl derivative of ACTH (NPS-ACTH) was shown to be a specific inhibitor of ACTH induced stimulation of lipolysis in rat fat cells as well as adenylate cyclase in rat fat cell ghosts. In view of the role of cyclic AMP in mediating the action of ACTH in the rat adrenal gland, the effects on NPS-ACTH on the synthesis of cyclic AMP in adrenal cells are of considerable interest. The stimulation of cyclic AMP synthesis by ACTH and NPS-ACTH in isolated rat adrenal cells prelabeled with H3-adenine has been investigated and the results showed that whereas ACTH produces a very large increase in cyclic AMP levels, NPS-ACTH causes only a small increase in cyclic AMP formation. NPS-ACTH also inhibits the ACTH induced cyclic AMP synthesis effectively.